News Column

Patent Issued for Enzyme-Catalyzed Metal Deposition for the Enhanced Detection of Analytes of Interest

May 6, 2014



By a News Reporter-Staff News Editor at Life Science Weekly -- A patent by the inventors Bieniarz, Christopher (Tucson, AZ); Farrell, Michael (Tucson, AZ), filed on June 21, 2012, was published online on April 22, 2014, according to news reporting originating from Alexandria, Virginia, by NewsRx correspondents (see also Ventana Medical Systems, Inc.).

Patent number 8703417 is assigned to Ventana Medical Systems, Inc. (Tucson, AZ).

The following quote was obtained by the news editors from the background information supplied by the inventors: "The present invention relates generally to the field of chemistry, and in particular, immunochemistry. More specifically, the present invention relates to a method for the enhanced detection of an analyte by using an enzyme-catalyzed reaction for the localized deposition of metal atoms. The present method of detection can be employed in various types of assays, such as, e.g., in-situ histochemical, immunoassays such as ELISA, and hybridization to nucleic acid micro arrays.

"Tissue staining is an ancient art by modern standards that goes back over one hundred years. Recently, efforts have been made to automate the procedure of applying different types of chemical and biochemical stains to tissue sections. Instruments that have been invented for this purpose include the Ventana Medical Systems' line of dual carousel-based instruments such as the 320, ES.RTM., NEXES.RTM., BENCHMARK.RTM., and the BENCHMARK.RTM. XT instruments. Patents that describe these systems include U.S. Pat. Nos. 5,595,707, 5,654,199, 6,093,574, and 6,296,809, all of which are incorporated herein by reference in their entirety. Another type of automated stainer is the TECHMATE.RTM. line of stainers, described in U.S. Pat. Nos. 5,355,439 and 5,737,499, both of which are incorporated herein by reference in their entireties.

"Over the years, various manual detection methods have been used in the field of histochemistry. Generally, once a molecular marker or target of interest in a tissue sample has been identified through biomolecular studies, it needs to be rendered visible under the light microscope for a pathologist or other medical specialist to interpret. The first detection step involves the use of an anti-target primary antibody detectably labeled with biotin, digoxigenin, fluorescein or other haptens to locate the biological target of interest. Next, an anti-hapten secondary antibody conjugated to an enzyme or other reporter molecule is used to locate the primary antibody. Typically used enzymes are well-known to those of ordinary skill in the art, and include horseradish peroxidase ('HRP') or alkaline phosphatase ('AP'). These enzymes then catalyze the precipitation of a chromogenic substrate in the immediate vicinity of the primary-secondary antibody complex. Chromogens such as nitro blue tetrazolium (NBT/BCIP), 3,3'-diaminobenzidine tetrahydrochloride (DAB), and 3-amino-9-ethylcarbazole (AEC) are well-known. Alternately, enzyme substrate interactions may produce chemiluminescent signals, which can be captured on photographic film.

"Other labels that have been used for histochemical detection include: .sup.125I-labeling of the secondary antibody, which can be detected using a photographic film; fluorescein isothiocyanate-labeled second antibody, which can be detected using UV light; .sup.125I-labeled Protein A, which can be used instead of a secondary antibody, as it will bind to the Fc region of IgG molecules; gold-labeled secondary antibody, which is directly visible as a red color when it is bound with the secondary antibody to the primary antibody; biotinylated secondary antibody, which when incubated with the secondary antibody, then incubated with enzyme-conjugated avidin or streptavidin ('SA') which binds strongly to the biotin, will give an enhanced signal, as multiple biotin molecules can be attached to a single antibody molecule. Enzymes typically used include AP or HRP.

"Enzyme immunoassays (EIA), such as, for example, enzyme-linked immunosorbant assays (ELISAs) are widely used for the determination, either qualitative or, mostly, quantitative, of a nearly unlimited variety of organic substances, either of natural origin or synthetic chemical compounds, such as peptides, proteins, enzymes, hormones, vitamins, drugs, carbohydrates, etc., for various purposes, such as, e.g., diagnostic, forensic, pharmacologic, and food quality control.

"Many different variants of ELISA methods exist that are familiar to those of ordinary skill in the art. In a classical 'sandwich' ELISA, to detect the presence or measure the concentration of an analyte (i.e., antigen) of interest such as tumor markers, hormones and serum proteins in a biological sample (e.g., blood, plasma, serum, urine, saliva, sputum), the sample is incubated on a solid support (e.g., microtiter well) that has been precoated with a first binding partner for the analyte (a.k.a. 'primary antibody' or 'capture antibody'). The sample and the primary antibody are incubated for a sufficient time to permit binding between the antibody and the antigen in the sample.

"As a result of this first reaction, any analyte present in the sample will have become bound to its binding partner and thereby to the solid support. After the solid support has been washed, steps are taken to make the result detectable. The solid support having attached thereto the binding partner and analyte, if any, is contacted with a second binding partner for the analyte (a.k.a. the 'secondary antibody'). In most cases, the secondary antibody carries a label/marker allowing its detection by generation of a chromogenic, fluorogenic, or other type of signal. In this type of assay, the antigen is 'sandwiched' in between the primary and the labeled secondary antibody.

"Typically, in ELISA-type assays, the label consists of an enzyme capable of a detectable conversion of a substrate, e.g., horseradish peroxidase is capable of converting, in the presence of hydrogen peroxide, a substrate, such as 3,3',5,5'-tetramethylbenzidine, into a colored product. Normally, after the enzyme-labeled reactant has been attached to the immobilized complex, the solid phase with complex bound thereto is washed before the actual detection phase is entered.

"In the detection phase, substrate solution is added to the solid phase with attached complex and the conversion, if any, of the substrate is detected. To allow quantitative measurement of the analyte, the solid phase is incubated with the substrate solution for a fixed time, which should be sufficiently long to allow a substantial enzymatic conversion of the substrate into a colored substance. After termination of the substrate-converting reaction, the intensity of the coloration, which is proportional to the immobilized amount of enzyme, is measured by optical means, such as a photometer to measure the absorbance at a chosen wavelength, such as 450 nm.

"A disadvantage of the existing ELISA techniques, however, is that the adsorption and detection phases, to secure assay sensitivity, can be very time consuming for samples having low analyte concentrations. In such samples, the rate of adsorption of analyte to the surface is proportional to the concentration of analyte in the solution, and thus will also become very low, even in well-stirred systems. To allow a reliable measurement of analytes present in a liquid at a concentration on the order of nanograms or even picograms per ml, the adsorption phase may require a reaction time of one to several hours.

"The most sensitive of such sandwich-type ELISA assays are capable of detecting 1 amol (1.times.10.sup.-18 M) of analyte. However, in the sandwich type ELISA, the analyte must have at least two antigenic sites that can be recognized simultaneously by the primary and secondary antibodies (or binding partners). Accordingly, such sandwich assays cannot be used for the detection of small peptides, most drugs, or synthetic drug candidates.

"Competitive ELISA is another type of ELISA methodology that is used to detect or quantify analytes such as small molecule antigens (i.e., T3, T4, progesterone, etc.). In competitive ELISA, a carefully titrated concentration of analyte-specific antibody is coated onto the inside wall of the microwell. In a single reaction, antigen from the test sample and the enzyme-labeled antigen conjugate compete for a limited number of immobilized antibody-binding sites. The amount of antibody-antigen-enzyme complex bound to the solid phase (microwell) is inversely proportional to the amount of antigen present in the sample.

"An expanding area of polynucleotide analysis is DNA array technology. This technology uses arrays of nucleic acid probes, such as oligonucleotides, to detect complementary nucleic acid sequences in a sample nucleic acid of interest (the 'target' nucleic acid). For example, an array of nucleic acid probes is fabricated at known locations on a substrate such as a chip or glass slide. A labeled nucleic acid is then brought into contact with the chip and a scanner generates an image file indicating the locations where the labeled nucleic acids are bound to the chip. Based upon the image file and identities of the probes at specific locations, it becomes possible to extract information such as the expression pattern of a nucleic acid of interest (see, e.g., U.S. Pat. No. 6,225,077).

"Methods using arrays of nucleic acids immobilized on a solid substrate are disclosed, for example, in U.S. Pat. No. 5,510,270. In this method, an array of diverse nucleic acids is formed on a substrate. The fabrication of arrays of polymers, such as nucleic acids, on a solid substrate, and methods of use of the arrays in different assays, are described in: U.S. Pat. Nos. 6,600,031, 6,576,424, 6,203,989, 6,180,351, 6,156,501, 6,083,726, 5,981,185, 5,744,101, 5,677,195, 5,624,711, 5,599,695, 5,445,934, 5,384,261, 5,571,639, 5,451,683, 5,424,186, 5,412,087, 5,384,261, 5,252,743 and 5,143,854; PCT WO 92/10092; PCT WO 93/09668; PCT WO 97/10365, which are incorporated by reference in their entireties.

"Accessing genetic information using high density DNA arrays is further described in Chee, Science 274:610-614 (1996). The combination of photolithographic and fabrication techniques allows each probe sequence to occupy a very small site on the support. The site may be as small as a few microns or even a small molecule. Such probe arrays may be of the type known as Very Large Scale Immobilized Polymer Synthesis (VLSIPS.TM.). U.S. Pat. Nos. 5,631,734 and 5,143,854 and PCT patent publication Nos. WO 90/15070 and 92/10092.

"Typically, the existence of a nucleic acid of interest in array technology and other DNA detection methods is indicated by the presence or absence of an observable 'label' attached to a probe or attached to amplified sample DNA.

"Clearly, whether one is detecting and/or quantifying analytes of interest in in situ histochemical assays, immunoassays, such as ELISA, or hybridization assays to arrays or microarrays of nucleic acids, detecting the signal formed by the particular enzymatic reaction sensitively, rapidly, and then being able to accurately relate it to the amount of analyte in a biological sample, is of paramount importance.

"Metallographic detection of analytes is well-known to those skilled in the arts of pathology, microscopy, and medical morphology. See, e.g., 'Gold and Silver Staining: Techniques in Molecular Morphology, edited by Gerhard W. Hacker and Jiang Gu, CRC Press, Boca Raton, Fla. (2002).

"Metallic enhancement of immunohistochemical detection is taught in U.S. Pat. No. 5,116,734 (Higgs et al.), incorporated herein by reference in its entirety. The '734 patent is directed to a composition of matter and a process for detecting the presence of an oxidative catalyst in a biological sample. The composition comprises a precipitate formed by oxidation of a chromogenic substrate in the presence of the catalyst, together with two or more co-precipitated reduced metals. A strong signal is formed with which to detect an oxidation catalyst which is localized to a target molecule. Target molecules may be nucleic acids, antibodies, or cell surface antigens. In particular, Higgs et al. rely on a chromogenic precipitate and two or more metals, for the purpose of detecting an oxidative catalyst.

"Merchanthaler et al., J. Histoch. And Cytochem., 37:1563-1565 (1989) teach silver intensification of the oxidatively polymerized chromogen DAB by pre-treating the DAB with nickel ions.

"A more recent example of metallic enhancement of immunohistochemical detection includes U.S. Pat. No. 6,670,113 (Hainfeld). The '113 patent is directed to a method of producing metal in a zero oxidation state from metal ions, comprising: providing metal ions of at least one metal selected from cesium, periodic table group 1b, 2a, 4a and 8, an oxygen containing oxidizing agent and a reducing agent selected from at least one of hydroquinone, a hydroquinone derivative or n-propyl gallate; providing an oxido-reductase enzyme; combining the enzyme with the metal ions, oxidizing agent and reducing agent; and reducing at least some of the metal ions to metal in a zero oxidation state. In particular, silver ion reduction to silver metal in proximity to horseradish peroxidase when exposed to hydrogen peroxide and hydroquinone is taught.

"There continues to be a need in the art for better and more sensitive biochemical techniques for detecting analytes of interest in various assay settings. For example, there is a need for improved methods of detecting immunohistochemical epitopes and DNA targets of interest via bright field light microscopy. There continues to be a need for more sensitive and improved methods for detecting and quantifying analytes of interest, such as antigens, cells, hormones, drugs, or any biologically active molecule, using enzyme immunoassays and more particularly, ELISA-based assays. Finally, there is a need in the art for better techniques for rapidly and sensitively detecting hybridization to arrays or microarrays of nucleic acids on solid supports."

In addition to the background information obtained for this patent, NewsRx journalists also obtained the inventors' summary information for this patent: "In the present invention, regardless of assay employed, the detection and quantification of the biological analyte of interest is based on a metal precipitate forming system that is catalyzed by an enzyme, such as, e.g., alkaline phosphatase. The enzyme catalyzes the dephosphorylation of a reducing agent precursor into an active reducing agent, which in turn reduces a metal ion into a detectable and quantifiable metal precipitate. By using the methods of the invention, low concentrations of analyte in assays such as in situ histochemical assays, immunoassays such as ELISA, or hybridization to DNA microarrays on glass slides, can be determined accurately, rapidly, and with high assay sensitivity.

"The present invention provides methods of detecting in situ an immunohistochemical epitope or nucleic acid sequence of interest in a biological sample comprising binding an enzyme-labeled conjugate molecule to the epitope or sequence of interest in the presence of a redox-inactive reductive species and a soluble metal ion, thereby catalyzing the reduction of the metal ion to a metal in oxidation state 0, at or about the point where the enzyme is anchored.

"The present invention also provides novel compositions of matter, which may be used in the claimed methods. Novel phosphate derivatives of reducing agents are described that when exposed to a phosphatase enzyme are activated to their reducing form, thereby reducing metal ions to insoluble metal particles in oxidation state 0.

"In one embodiment, the present invention provides a compound having the general structure (IV) shown below:

"##STR00001##

"wherein

"R.sub.1 may be H, alkyl, aryl, carboxyl, carboxyalkyl, NH.sub.2, (CH.sub.2).sub.n--COOH--, nitro, ether, thioether and sulphonate;

"R.sub.2 may be H, alkyl, aryl, carboxyl, carboxyalkyl, NH2, or (CH.sub.2).sub.n--COOH--, nitro, ether, thioether and sulphonate; and

"Z may be PO.sub.3.sup.2-, H, .alpha.-galactose, .beta.-galactose, .alpha.-glucose, .beta.-glucose, ester, and .beta.-lactam; but both Zs may not be H. A preferred compound has both Zs=phosphate.

"In another embodiment, the present invention is directed to a compound having the general structure (V):

"##STR00002##

"wherein

"R.sub.1 may be H, alkyl, aryl, carboxyl, carboxyalkyl, NH2, (CH.sub.2).sub.n--COOH--, nitro, ether, thioether and sulphonate; and

"Z may be PO.sub.3.sup.2-, .alpha.-galactose, .beta.-galactose, .alpha.-glucose, .beta.-glucose, ester, and .beta.-lactam. A particularly preferred compound has Z=phosphate.

"In yet another embodiment, the present invention is directed to a compound having the general structure (VI):

"##STR00003##

"wherein

"R.sub.1 may be H, alkyl, aryl, carboxyl, carboxyalkyl, NH2, (CH.sub.2).sub.n--COOH--, nitro, ether, thioether or sulphonate; and

"Z may be PO.sub.3.sup.2, .alpha.-galactose, .beta.-galactose, .alpha.-glucose, .beta.-glucose, ester, and .beta.-lactam. A particularly preferred compound has Z=phosphate.

"In yet another embodiment, the present invention is directed to a compound having the general formula (VII):

"##STR00004##

"wherein

"R.sub.1 may be H, alkyl, aryl, carboxyl, carboxyalkyl, NH2, (CH.sub.2).sub.n--COOH--, nitro, ether, thioether and sulphonate; and

"Z may be PO.sub.3.sup.2, .alpha.-galactose, .beta.-galactose, .alpha.-glucose, .beta.-glucose, ester, and .beta.-lactam. Two particularly preferred compounds have R.sub.1=methyl or CH.sub.2--(CH.sub.2--CH.sub.2--CH(CH.sub.3)--CH.sub.2).sub.3--H, and Z=phosphate.

"The present invention also provides a method of in situ staining a biological sample having an epitope or nucleotide sequence of interest, comprising the steps of:

"(a) contacting said biological sample with a conjugate molecule having a hapten; (b) contacting said hapten with a hapten-binding partner conjugated to a label enzyme; and © contacting said biological sample with a redox-inactive reductive species that is a substrate for said label enzyme in the presence of a metal ion.

"The present invention also provides a method of in situ staining a biological sample having an epitope or nucleotide sequence of interest, comprising the steps of:

"(a) contacting said biological sample with a biotinylated primary antibody; (b) contacting said biological sample having said biotinylated primary antibody bound to it with a streptavidin-alkaline phosphatase conjugate; and © contacting said biological sample of step (b) with ascorbic acid phosphate in the presence of silver ion at a pH greater than 7. An optional gold ion pretreatment step may also be applied in order to enhance the silver deposition.

"The present invention also provides a method for detecting and quantifying an analyte of interest in a biological sample using an enzyme immunoassay, such as an ELISA-based assay, by measuring enzyme-catalyzed metal precipitate formation on the surface of the solid support. More specifically, the method comprises the steps of: (a) contacting a biological sample containing an analyte of interest with a primary antibody immobilized on a solid support, such that the presence of the analyte in the biological sample will result in the formation of a primary antibody-analyte complex; (b) contacting said complex of step (a) with a secondary antibody conjugated to a label enzyme, such that the presence of analyte in the biological sample will result in a primary antibody-analyte-secondary antibody complex; © contacting the complex formed in step (b) with a redox inactive reductive species in the presence of metal ion so that a metal precipitate will form on the solid support following reduction; (d) enhancing said metal precipitate; and (e) detecting and quantifying the amount of analyte in the sample by measuring the amount of metal precipitate formed on the solid support.

"A particularly preferred enzyme label is alkaline phosphatase, a particularly preferred redox inactive reductive species is ascorbic acid phosphate, and a particularly preferred metal ion is silver ion. An optional gold ion pretreatment step may also be applied to enhance the silver deposition. In the embodiment where the enzyme label is alkaline phosphatase, the optimal pH is between 7 and 10.5, preferably 8-9, and most preferably 9.

"The present invention also provides a method for detecting nucleic acid sequences following hybridization to nucleic acid microarrays by measuring enzyme-catalyzed metal precipitate formation on the surface of the solid support immobilizing the nucleic acid. More specifically, the method comprises the steps of: (a) labeling DNA isolated from a biological sample with a first member of a specific binding pair; (b) hybridizing said DNA of step (a) to a microarray of nucleic acid sequences immobilized on a solid support under appropriate hybridization conditions; © incubating said microarray of step (b) with a label enzyme conjugated to a second member of said specific binding pair; (d) incubating said microarray with a redox inactive reductive species in the presence of metal ion so that a metal precipitate will form on the microarray following reduction; (e) enhancing said metal precipitate; and (f) detecting nucleic acid sequences that are complementary to at least one of the microarray of nucleic acid sequences immobilized on the solid support by visualizing the metal precipitate formed on the solid support.

"The present invention also provides kits for carrying out the claimed methods conveniently.

"In any of the methods or kits of the invention, the detection and quantification of the biological analyte of interest is based on a metal precipitate forming system that is catalyzed by an enzyme, such as, e.g., alkaline phosphatase. The enzyme catalyzes the dephosphorylation of a redox inactive reductive species into an active reducing agent, which in turn reduces metal ion into a detectable and quantifiable metal precipitate.

"By using the methods of the invention, low concentrations of analyte in either an in situ histochemical assay, an immunoassay, such as ELISA, or following nucleic acid hybridization to ordered DNA microarrays on a glass slide, can be determined rapidly. Thus, the present invention results in a large increase in assay sensitivity in shorter assay times."

URL and more information on this patent, see: Bieniarz, Christopher; Farrell, Michael. Enzyme-Catalyzed Metal Deposition for the Enhanced Detection of Analytes of Interest. U.S. Patent Number 8703417, filed June 21, 2012, and published online on April 22, 2014. Patent URL: http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&p=92&u=%2Fnetahtml%2FPTO%2Fsearch-bool.html&r=4554&f=G&l=50&co1=AND&d=PTXT&s1=20140422.PD.&OS=ISD/20140422&RS=ISD/20140422

Keywords for this news article include: Antibodies, Antigens, Anions, Phenols, Hormones, Chemistry, Immunology, Phosphates, Technology, Biochemical, Peroxidases, DNA Research, Legal Issues, beta-Lactams, Hydroquinones, Blood Proteins, Immunoglobulins, Oxidoreductases, Reducing Agents, Phosphoric Acids, Sulfur Compounds, Hydrogen Peroxide, Biological Factors.

Our reports deliver fact-based news of research and discoveries from around the world. Copyright 2014, NewsRx LLC


For more stories covering the world of technology, please see HispanicBusiness' Tech Channel



Source: Life Science Weekly


Story Tools






HispanicBusiness.com Facebook Linkedin Twitter RSS Feed Email Alerts & Newsletters